Screening Strains For Microbial Treatment To Produce High-purity Fructooligosaccharides,Optimization Of Its Cultivation And Purification Conditions And Investigation On Scale-up Process

Fructo-oligosaccharides（FOS）such as 1-kestose,nystose,and fructosyl-nystose are functional oligosaccharides produced by fructosyltransferase（β-fructofuranosidase）.They are used as food ingredients and have beneficial effects on health in humans and improve the growth of beneficial bacteria such as bifidobacteria in intestinal flora.However,limited by the inhibitory effect of the substrate,fructo-oligosaccharide mixtures produced by fermentation contain large amounts of non-prebiotic sugars,such as unreacted sucrose,glucose and fructose.Since high-purity FOS with decreased amounts of monosaccharides and sucrose are desirable for some uses,such as for products for consumers with diabetes mellitus,strategies have been developed for obtaining FOS with high purity.Numerous studies have been focused on the development of downstream treatments to purify FOS from sugar mixtures,such as the use of membrane techniques,chromatographic methods and microbial treatment using successive purification fermentation.However,traditional physical and biochemical separation methods are considered to be expensive and costly,and are not conducive to wide application and promotion.Therefore,microbial treatment is considered to be a feasible and economical alternative method.At present,little research has been done on the microbial treatment,and the purification results are generally poor,which makes this study appear more important.In this study,a one-stage process of microbial treatment for simultaneous production and purification of FOS was established.A strategy for screening strain based on the glucose plate assay and FOS plate assay was first developed,and a stable genetically purified FOS strain was screened from the natural environment.The strain of GXL-22 was identified as Wickerhamomyces anomalus,based on colony morphology observation,cell morphology observation,physiological and biochemical test,and 18S r DNA sequence analysis.The samples were analyzed by high-performance liquid chromatography（HPLC）to verify the ability of strain GXL-22 to remove residual sugars in FOS-50.The results showed that strain GXL-22 could effectively removed glucose and fructose in FOS within 24 hours,and the purity of FOS after purification was 82.4%（w/w）.In order to obtain a large number of yeast cells suitable for industrial production,the culture conditions and culture medium components of strain GXL-22 in shake-flask were optimized by single factor experiments and orthogonal experiments.The optimum culture medium components was determined as follows:glucose 40 g/L,wort 4%（w/v）,yeast extract 10 g/L,Mn SO₄0.4 g/L.The optimum culture conditions were determined as follows:inoculation volume of 6%（v/v）,loaded liquied of 50 m L in 500 m L flask,culture temperature of 28℃,p H 5.5,shaking speed of 240 rpm,culture time of 48 h.After optimization,the OD₆₆₀of the strain GXL-22 in the shake flask reached0.861 at 48 h,and the dry weight of the bacterial cell reached 20.95 g/L,which was 68.8%and 83.8%higher than the initial conditions,respectively.Taking the shake flask results as a reference,a scale-up experiment was carried out in a 5 L fermenter,and the rotate speed and aeration volume were optimized.After optimization,the OD₆₆₀of the strain GXL-22 in the fermenter reached 0.951 at40 h,and the dry weight of the bacteria reached 24.42 g/L,which was 10.5%and 16.6%higher than the shake flask results,respectively.The conditions for the production of high-purity FOS from strain GXL-22 in shake-flask were optimized by single factor experiments and orthogonal experiments.The optimum purification conditions was determined as follows:25°BX FOS as the substrate,p H 6.5,temperature of 30℃,shaking speed of 120 rpm,inoculation volume of 20%（v/v）,purification time of 24 h.The highest purity of FOS reached 87.2%,but there were still some sucrose that could not be effectively removed.In order to reduce the sucrose and obtain higher purity FOS,the purification processes of high-purity FOS production by GXL-22 were studied.The content of sucrose in the FOS mixture could be reduced by optimized the transglycosylation time of FOS and addition of fructosyltransferase.The highest purity of FOS reached 90.4%was obtained after optimized transglycosylation time;and the highest purity of FOS reached 91.6%was obtained by hydrolyzing sucrose with fructosyltransferase.The purification process of high-purity FOS production with FOS-50 and sucrose as substrate was established in the 10-L fermentor,respectively.Under the optimized fermentation conditions,the highest purity of FOS reached 93.2%with FOS-50 as a substrate.In order to reduce the loss of substrate and simplify the process step,an integrated process enabling the simultaneous production and purification of FOS was established.The highest purity of FOS reached 95.6%at 52 h with sucrose as a substrate,and the recovery reached 86.9%.The results provided some ideas and value references for the industrial production of high-purity FOS.